Process for selecting anti-angiogenesis antibody fragments

Description

Field of the invention

[0001] The present invention relates to a process for filter selection of anti-angiogenesis antibody fragments from a large combinatorial repertoir; the invention further relates to the anti-angiogenesis antibody fragments thus obtained.

State of the art

[0002] As is generally known, functional antibody fragments can be produced in Escherichia

[0003] Coli and displayed on phages, and the processes for selecting antibodies from phage-displayed libraries have now become routine methods allowing to obtain fragments of monoclonal antibodies which can interact practically with any type of antigen in a simple and fast way, at least when the purified antigen is available.

[0004] For example, Pini et al J. Biol Chem (1998) 273 34 21769-21776 reports the construction and use of a phage display library to select antibodies against six biologically relevant antigens, including the ED-B domain of fibronectin and Borsi et al Exp Cell Res (1998) 240 244-251 reports the selection of human antibody fragments to the ED-A domain of fibronectin from a phage display library and their subsequent characterisation.

[0005] It is also known that other techniques allow to avoid the use of phages thanks to the paper selection of antibody fragments secreted by individual bacterial clones.

[0006] In particular, Skerra et al. [Anal. Biochem. 196, 151-155 (1991)] have described a two-membrane system which can determine the bond of an antigen to Fab antibody fragments secreted by bacterial colonies. In short, the method consists in growing bacterial colonies expressing antibodies on a first porous filter and trapping the secreted fragments on another membrane which is then tested for its bond with the antigen. The clones expressing binding antibodies can thus be identified on the first filter and re-grown. However, the method described has been carried out in simple systems consisting of two different binding specificities and starting from few thousands of bacteria, obviously non confluent.

[0007] It is obviously quite important to have methods enabling the selection of specific binders starting from large repertoires of antibody fragments expressed in bacteria.

[0008] As a matter of fact, such methods would allow to avoid the display of antibodies on phages and to identify directly the clones which can express antibody fragments in soluble form.

Summary of the Invention

[0009] The present invention relates to a process as defined in claim 1 in which starting from a highly complex system containing billions of different binding specificities, several cycles of filter selection of colonies and of amplification of positive clones are carried out.

Description of the Figures

[0010] 

Figure 1: it shows schematically the method according to the invention as hereinafter described.

Figure 2: it shows the deposit membrane (A) and the trap membrane (B) after the development in the third screening cycle of colonies binding to ED-B.
As is evident at this stage, the colonies on the deposit membrane are not confluent anymore, about 30% of the colonies on (A) have produced antibody fragments scFVS which can bind to (B), each of which is therefore a source of anti-ED-B monoclonal antibody fragments.
Figure 3: it shows on the right the bond of the antibody ME-4C to each synthetic decapeptide of ED-B sequence and represents it on a horizontal line. The thickness of said line is proportional to the binding affinity observed. Two minimum sequences which have been recognized by the antibodies within the group of synthetic peptides ED-B are in cyan and magenta. The same colors are used on the left-hand side of the figure to indicate the position of these two epitopes on the three-dimensional structure of ED-B.
Figure 4: it summarizes the results of immunohistochemical tests carried out with the antibody fragment scFv ME-4C.

(A) is a section.of the sample of a multiform glioblastoma. The typical glomerulus-shaped vascular structures are colored in red by scFv ME -4C.

(B) is a section of human melanoma SKMEL-28 colored with the antibody fragment scFv ME-4C. The antibody places itself around vascular structures and proliferating cells.

Detailed description of the invention

[0011] In the process according to the present invention bacteria containing the DNA encoding several binding specificities are grown in a liquid medium and, once the stage of exponential growth has been reached, some billions of them are distributed onto a growth support consisting of a suitably pre-treated membrane filter which is incubated until completely confluent bacteriae colonies appear.

[0012] A second trap substrate consists of another membrane filter, pre-humidified and covered with the desired antigen.

[0013] The trap membrane filter is then placed onto a plate containing a suitable culture medium and covered with the growth filter with the surface covered with bacterial colonies pointing upwards. The sandwich thus obtained is incubated at room temperature for about 16 h. It is thus possible to obtain the expression of the genes encoding antibody fragments scFv having a spreading action, so that those fragments binding specifically with the antigen which is present on the trap membrane are trapped.

[0014] The trap membrane is then treated to point out bound antibody fragments scFv with colorimetric techniques commonly used to this purpose.

[0015] The position of the colored spots on the trap filter allows to go back to the corresponding bacterial colonies which are present on the growth membrane and produced the antibody fragments trapped.

[0016] Such colonies are gathered and grown and the bacteria - a few millions of them - are distributed onto a new culture membrane repeating the procedures described above.

[0017] Analogous cycles are then carried out until the positive signals on the trap membrane correspond to single positive colonies, each of which represents a potential source of monoclonal antibody fragments directed against the antigen used in the selection.

[0018] In particular, the process according to the invention may be carried out with bacteria of E.coli, strain TG1, hosting DNA encoding the various binding specificities and containing ETH-2 library [described in Viti et. al. Methods in Enzymology, 326, 480-505]. As trap antigen the recombinant protein 7889 has been chosen [Castellani et al. (1994) Int. J. Canc. 59, 612-618] containing the domains 7, ED-B, 8, 9 of Fibronectin.

[0019] By operating as described above the clones obtained at the end of the third cycle are grown and produce soluble fragments of antibody fragments scFvs.

[0020] In particular, one of the monoclonal antibody fragments (hereinafter identified as ME-4C) is isolated and sequenced thanks to its capacity of binding to ED-B of Fibronectin. ME-4C binds to ED-B with an affinity in the order of nanomoles according to the data obtained for antibody fragments previously selected from the same repertoir.

Experimental section

ETH-2 library

[0021] This is a phage-display library of recombinant antibody fragments scFv containing more than 5 x 10⁸ individual clones. The diversity is introduced into the antibodies in the Complementarity Determining Region 3 (CDR-3) both of the Variable Heavy (VH) chain and of the Variable Light (VL) chain. The genes encoding VH and VL are cloned in a vector enabling their expression in soluble form.

[0022] Moreover, said vector introduces an identification on the carboxylic terminal of antibody fragments scFv.

Growth of the colony and expression of antibody fragments scFv

[0023] ETH-2 library is kept as bacteria (E.coli, strain TG1) hosting the DNA encoding the various specificities. Said bacteria are grown in a liquid medium and once they have reached the stage of exponential growth 10⁸, they are distributed on a Durapore membrane filter in PVDF with a diameter of 20 cm (type GVWP, Millipore). Said filter is placed in a Petri plate with a diameter of 20 cm, containing TYE Agar (Miller, 1972), 100 µg/ml of Ampicillin, 1 % of glucose, and incubated at 37°C for 8 h. After the incubation completely confluent bacteriae colonies can be seen on the filter.

[0024] A second (trap) membrane is prepared by pre-humidifying a membrane with a diameter of 20 cm in PVDF (Immobilon-P, Millipore) and covering it with the trap antigen. As trap antigen the recombinant protein 7B89 containing domains 7, ED-B, 8, 9 of Fibronectin is chosen. The recombinant domain ED-B of Fibronectin alone, i.e. our target for antibody isolation, does not bind to the membrane. The covering of the membrane is obtained by incubating said membrane at 37°C for 6 h in a solution of 50 mM phosphate, pH 7.4, 100 mM NaCl (phosphate buffer, PBS) containing 150 µg/ml of 7B89. The filter is then blocked in a 5% solution milk/PBS (MPBS) at 37°C for 2 h, washed 4 times in 0.2% PBS (v/v) Tween 20 (PBST) and immersed in 2xTY containing 100 µg/ml of Ampicillin and 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG).

[0025] The trap membrane is placed on a plate of TYE Agar containing 100 µg/ml of Ampicillin and 1 mM of IPTG and covered with Durapore membrane with the bacterial colonies pointing upwards.

[0026] The sandwich structure thus obtained is incubated at room temperature for 16 hours. The presence of IPTG in the medium together with the absence of glucose enables the expression (regulated by Lac promoter) of the genes encoding antibody fragments scFvs having a spreading action, those specifically binding for protein 7B89 being thus trapped by the second membrane.

Detection of the bond with the antigen

[0027] The trap membrane is washed four times with PBST and blocked in MPBS at 37°C for 6 h. In order to determine the bound antibody fragments scFv, the filter is incubated with anti-flag M2 murine antibody (Sigma, F-3165) diluted 1:3000 in MBST at 37°C for 1 h, washed 4 times in PBST and incubated with Horseradish Peroxidase (HRP) conjugated with anti-mouse rabbit IgG (bioRad, 172-1011) diluted 1:3000 in MBST at 37°C for 1 h. The filter is then abundantly washed with PBST and incubated in 4-chloro-1-naphthol until dark spots are observed. The enzymatic colorimetric reaction is interrupted by washing with water and the filter is then dried.

[0028] The alignment signs obtained by placing the two filter one onto the other allows to go back from the colored spots on the trap filter to the producing bacterial colony of the first filter.

Amplification of positive clones

[0029] After the first screening cycle the positive signals on the trap filter correspond to the areas of confluent bacteriae colonies on the first filter which have been gathered and grown in 2xTY, 100 µg/ml of Ampicillin, 1% (w/v) of glucose up to O.D. (600 nm) = 0.7.

[0030] At this stage 10⁶ bacteria are distributed onto a new Durapore membrane and the processes described above are repeated. At the end of the second cycle the positive signals correspond to monocoalescent colonies. 18 of these colonies are picked up, grown and used for a successive screening in which 5 x10³ bacteria are placed on the Durapore filter. After the third cycle the positive signals on the trap membrane correspond to single positive colonies, each, of which represents a source of monoclonal antibodies directed against antigen 7B89.

Characterization of selected antibodies

[0031] Individual positive clones obtained after the third screening cycle as described above are grown in a liquid medium and produce soluble fragments of antibody fragments scFvs. The binding specificity of these fragments is then determined with an ELISA test carried out on ED-B, 7B89 or irrelevant antigens (MPBS, BSA, Ovalbumin, Lysoenzyme).

[0032] The kinetic coefficients concerning association and dissociation of antibody fragments are determined by plasma surface resonance with BIAcore 1000 (Pharmacia) following known protocols. Few clones which were positive to the ELISA test are chosen for the measurements. The measurements are carried out with the monomer fraction of antibody fragments scFvs as described in relevant documents, using chips covered both with ED-B and with 7B89.

[0033] Clone sequencing is carried out with a DNA sequencer 377 ABI Prism (Perkin-Elmer). The epitopes on ED-B recognized by one of the antibody fragments scFvs selected (hereinafter defined as ME-4C) are identified using the SPOT synthesis method as described for other anti-ED-B antibody fragments.

Immunohistochemistry

[0034] The immunocoloring of sections of multiform glioblastoma and human melanoma SKMEL is carried out as known in the corresponding scientific literature.

[0035] In order to identify whether the antibody isolated from the library with the process described above had a good affinity towards the native antigen, a real-time interaction analysis is applied using plasma surface resonance with a BlAcore device. The monomer fraction of ME-4C binds to ED-B with an affinity of 10^-7 M^-1 with a kinetic dissociation constant (K_off) of 6 x 10^-3 s^-1 and a kinetic association constant (K_on) of 6 x 10⁴ s^-1 M^-1.

[0036] In order to further characterize the bond between the antibody fragment ME-4C and ED-B, the epitopes recognized by the antibody fragment scFv are identified using the SPOT synthesis method. A series of 82 decapeptides covering the sequences of the 91 ED-B residues is tested for its bond with ME-4C.

[0037] In order to determine whether the selected antibody fragment scFv ME-4C could act as reagent in the identification of angiogenesis in tissue samples, studies are carried out on tumor sections: the results are summarized in Figure 4.

[0038] As shown by immunohistochemical experiments carried out on sections of human multiform glioblastoma and melanoma, the antibody ME-4C can mark blood vessels building up in tumor tissues, which makes said antibody a valuable help for clinical applications such as the analysis of tissue sections for the angiogenesis determination.

SEQUENCE LISTING

[0039] 

<110> PHILOGEN s.r.1.

<120> Process for selecting anti-angiogenesis antibody fragments, antiangiogenesis antibody fragments thus obtained and their use

<130> not filed yet

<150> FI2000A000247
<151> 2000-12-06

<160> 4

<170> PatentIn version 3.1

<210> 1
<211> 720
<212> DNA
<213> homo sapiens

<220>
<221> CDS
<222> (1)..(720)
<223> nucleotide sequence corresponding to scFv ME-4C

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<210> 2
<211> 240
<212> PRT
<213> homo sapiens

<400> 2

<210> 3
<211> 8
<212> PRT
<213> homo sapiens

<220>
<221> DOMAIN
<222> (1)..(8)
<223> region corresponding to the CDR3 of ME-4C scFv heavy chain

<400> 3

<210> 4
<211> 10
<212> PRT
<213> homo sapiens

<220>
<221> DOMAIN
<222> (1)..(10)
<223> region corresponding to CDR3 of ME-4C scFv light chain

<400> 4

Claims

1. Process for selecting anti-angiogenesis antibody fragments, comprising;

(i) distributing bacterial colonies expressing billions of different binding specificities in soluble form onto a first membrane filter, wherein the binding specificities comprise antibody fragments,

(ii) incubating the filter until the colonies are confluent,

(iii) providing a second membrane filter comprising an immobilised antigen,

(iv) placing the second membrane filter in a plate containing culture medium,

(v) covering the second membrane filter with the first membrane filter with the bacterial colonies pointing upwards,

(vi) incubating the first and second membrane filters, such that antibody fragments expressed by the colonies on the first membrane filter bind to the antigen on the second membrane filter,

(vii) detecting the binding of antibody fragments to the second filter using colorimetric techniques,

(viii) identifying by the position of the colored spots on the second membrane filter the bacterial colonies on the first membrane filter which expressed the antibody fragments bound to the second membrane filter,

(ix) growing the identified bacterial colonies in liquid medium

(x) distributing said identified bacterial colonies onto a membrane filter and

(xi) repeating steps (iii) to (x) for additional cycles until single colonies expressing antibody fragments which bind to the second filter are identified in step (viii), said single colonies producing soluble anti-angiogenesis antibody fragments.

2. Process according to claim 1, in which at least three additional cycles are carried out.

3. Process according to claim 2, in which:

a) colonies of E. coli bacteria strain TG1 hosting the DNA encoding the various binding specificities and preserving an scFv library are distributed onto a first membrane filter (growth filter);

b) the antigen 7B89 containing the domains 7, ED-B, 8, 9 of Fibronectin is applied onto a second membrane filter (trap filter);

c) the trap filter is placed in a plate containing culture medium and covered with the growth filter with the bacterial colonies pointing upwards;

d) the positive signals due to the bond of the antibody fragments scFvs are identified on the trap filter by means of common colorimetric reactions;

e) the positions of the signals identified in step (d) are compared with the colonies which are present on the growth filter by picking up the colony areas corresponding to said signals;

f) the colonies gathered in step (e) undergo two further analogous growth and identification cycles;

g) the single colonies identified are picked up from the last cycle;

h) the scFv's produced by the colonies gathered in step (g) are picked up.

Ansprüche

1. Verfahren zur Auswahl von Anti-Angiogenese-Antikörper-Fragmenten, umfassend:

(i) das Verteilen bakterieller Kolonien, die Milliarden unterschiedlicher Bindungsspezifitäten in löslicher Form exprimieren, auf einem ersten Membranfilter, worin die Bindungsspezifitäten Antikörper-Fragmente umfassen,

(ii) das Inkubieren des Filters, bis die Kolonien konfluent sind,

(iii) das Bereitstellen eines zweiten Membranfilters, umfassend ein immobilisiertes Antigen,

(iv) das Platzieren des zweiten Membranfilters in einer Platte, die Kulturmedium enthält,

(v) das Bedecken des zweiten Membranfilters mit dem ersten Membranfilter, wobei die bakteriellen Kolonien nach oben zeigen,

(vi) das Inkubieren des ersten und zweiten Membranfilters, so dass Antikörperfragmente, die durch die Kolonien auf dem ersten Membranfilter exprimiert werden, an das Antigen auf dem zweiten Membranfilter binden,

(vii) das Detektieren der Bindung von Antikörperfragmenten an den zweiten Filter unter Verwendung kolorimetrischer Verfahren,

(viii) das Identifizieren der bakteriellen Kolonien auf dem ersten Membranfilter, die die Antikörperfragmente exprimierten, die an den zweiten Membranfilter gebunden sind, durch die Position der gefärbten Flecken auf dem zweiten Membranfilter,

(ix) das Züchten der identifizierten bakteriellen Kolonien in flüssigem Medium,

(x) das Verteilen der identifizierten bakteriellen Kolonien auf einem Membranfilter und

(xi) das Wiederholen der Schritte (iii) bis (x) für zusätzliche Zyklen, bis Einzelkolonien, die Antikörperfragmente exprimieren, die an den zweiten Filter binden, in Schritt (viii) identifiziert werden, wobei die Einzelkolonien lösliche Anti-Angiogenese-Antikörper-Fragmente produzieren.

2. Verfahren nach Anspruch 1, bei dem zumindest drei zusätzliche Zyklen durchgeführt werden.

3. Verfahren nach Anspruch 2, bei dem:

a) Kolonien von E.-coli-Bakterienstamm TG1, welche die DNA, die für die verschiedenen Bindungsspezifitäten kodiert, in sich tragen und eine scFv-Bibliothek konservieren, auf einen ersten Membranfilter (Wachstumsfilter) verteilt werden;

b) das Antigen 7B89, das die Domänen 7, ED-B, 8, 9 von Fibronectin enthält, auf einen zweiten Membranfilter (Einfangfilter) aufgebracht wird;

c) der Einfangfilter in einer Platte platziert wird, die Kulturmedium enthält, und mit dem Wachstumsfilter bedeckt wird, wobei die bakteriellen Kolonien nach oben zeigen;

d) die positiven Signal aufgrund der Bindung der Antikörper-Fragmente scFvs auf dem Einfangfilter mittels herkömmlicher kolorimetrischer Reaktionen identifiziert werden;

e) die Positionen der Signale, die in Schritt (d) identifiziert werden, mit den Kolonien verglichen werden, die auf dem Wachstumsfilter vorhanden sind, indem die Koloniebereiche herausgesucht werden, die den Signalen entsprechen;

f) die Kolonien, die in Schritt (e) gesammelt wurden, zwei weiteren analogen Wachstums- und Identifikationszyklen unterzogen werden;

g) die Einzelkolonien, die identifiziert wurden, vom letzten Zyklus herausgesucht werden;

h) die scFvs, die von den Kolonien produziert wurden, die in Schritt (g) gesammelt wurden, herausgesucht werden.

Revendications

1. Procédé de sélection de fragments d'anticorps anti-angiogenèse, comprenant les étapes consistant à :

(i) distribuer des colonies bactériennes exprimant des milliards de spécificités de liaison différentes sous forme soluble sur un premier filtre à membrane, dans lequel les spécificités de liaison comprennent des fragments d'anticorps,

(ii) incuber le filtre jusqu'à ce que les colonies soient confluentes,

(iii) fournir un second filtre à membrane comprenant un antigène immobilisé,

(iv) placer le second filtre à membrane dans une plaque contenant un milieu de culture,

(v) recouvrir le second filtre à membrane du premier filtre à membrane, les colonies bactériennes étant dirigées vers le haut,

(vi) incuber les premier et second filtres à membrane, de telle sorte que des fragments d'anticorps exprimés par les colonies sur le premier filtre à membrane se lient à l'antigène sur le second filtre à membrane,

(vii) détecter la liaison de fragments d'anticorps au second filtre en utilisant des techniques colorimétriques,

(viii) identifier par la position des taches colorées sur le second filtre à membrane les colonies bactériennes sur le premier filtre à membrane qui exprimaient les fragments d'anticorps liés au second filtre à membrane,

(ix) faire croître les colonies bactériennes identifiées dans le milieu liquide,

(x) distribuer lesdites colonies bactériennes identifiées sur un filtre à membrane et

(xi) répéter les étapes (iii) à (x) pour des cycles supplémentaires jusqu'à ce que des colonies uniques exprimant des fragments d'anticorps qui se lient au second filtre soient identifiées dans l'étape (viii), lesdites colonies uniques produisant des fragments d'anticorps anti-angiogenèse solubles.

2. Procédé selon la revendication 1, dans lequel au moins trois cycles supplémentaires sont effectués.

3. Procédé selon la revendication 2, dans lequel :

a) des colonies de souche de bactéries E. coli TG1 hébergeant l'ADN codant les diverses spécificités de liaison et conservant une bibliothèque de scFv sont distribuées sur un premier filtre à membrane (filtre de croissance) ;

b) l'antigène 7B89 contenant les domaines 7, ED-B, 8, 9 de fibronectine est appliqué sur un second filtre à membrane (filtre piège) ;

c) le filtre piège est placé dans une plaque contenant un milieu de culture et recouvert du filtre de croissance, les colonies bactériennes étant dirigées vers le haut ;

d) les signaux positifs dus à la liaison des fragments d'anticorps scFv sont identifiés sur le filtre piège au moyen de réactions colorimétriques communes ;

e) les positions des signaux identifiés dans l'étape (d) sont comparées aux colonies qui sont présentes sur le filtre de croissance en relevant les zones de colonie correspondant auxdits signaux ;

f) les colonies rassemblées dans l'étape e) subissent deux cycles de croissance et d'identification analogues supplémentaires ;

g) les colonies uniques identifiées sont relevées du dernier cycle ;

h) les scFv produits par les colonies rassemblées à l'étape g) sont relevés.

Drawing